Abstract

Anti-tumor-necrosis-factor-α (TNF-α) monoclonal antibody was used to treat Tg197 transgenic
mice, which constitutively produce human TNF-α (hTNF-α) and develop a progressive
polyarthritic disease. Treatment of both young (7- or 8-week-old) and aged (27- or
28-week-old) mice commenced when at least two limbs showed signs of moderate to severe
arthritis. The therapeutic efficacy of anti-TNF-α antibody was assessed using various
pathological indicators of disease progression. The clinical severity of arthritis
in Tg197 mice was significantly reduced after anti-TNF-α treatment in comparison with
saline-treated mice and in comparison with baseline assessments in both young and
aged mice. The treatment with anti-TNF-α prevented loss of body weight. Inflammatory
pathways as reflected by elevated circulating hTNF-α and local expression of various
proinflammatory mediators were all diminished by anti-TNF-α treatment, confirming
a critical role of hTNF-α in this model of progressive polyarthritis. More importantly,
the amelioration of the disease was associated with reversal of existing structural
damage, including synovitis and periosteal bone erosions evident on histology. Repair
of cartilage was age dependent: reversal of cartilage degradation after anti-TNF-α
treatment was observed in young mice but not in aged mice.

Keywords:

Introduction

Rheumatoid arthritis (RA) is a significant, chronic disease that afflicts 1% of the
general population in most countries [1]. Joint damage typically occurs before patients are diagnosed, and most of the joint
destruction occurs within the first 2 years of diagnosis [2]. Therapeutic drugs such as sulfasalazine and methotrexate (MTX) only slow the progression
of the disease, suggesting that these drugs fail to adequately quell the underlying
pathophysiology of RA [3].

Tumor necrosis factor alpha (TNF-α) is elevated in sera and synovial fluid of patients
with RA, suggesting that it may play a role in the pathology of the disease [4]. Soluble TNF-α receptor or neutralizing antibodies against TNF-α have been shown
to prevent collagen-induced arthritis in mice [5,6]. Furthermore, a human TNF-α (hTNF-α) transgenic mouse (Tg197) develops a chronic,
progressive polyarthritis with histologic features in common with RA [7]. Weight loss and joint swelling in these mice are correlated with expression of hTNF-α
mRNA in the joints [7] and hTNF-α concentrations in the serum [8,9]. Previously, Keffer et al.[7] showed that progression of disease in these mice could be prevented by treatment
with anti-TNF-α monoclonal antibody (mAb). In this study, we utilized the Tg197 mouse
model to further assess whether anti-TNF-α treatment can ameliorate established disease
in both young and aged mice.

Materials and methods

Tg197 mice and anti-TNF-α mAb treatment

Heterozygous Tg197 transgenic mice, obtained from Dr George Kollias (Hellenic Pasteur
Institute, Athens, Greece) [7], were identified by PCR analysis using primers to the 3'-modified hTNF-α gene. Two
experimental groups of 25 mice were used. 'Young mice' were enrolled into the study
when they developed clinical signs of arthritis at 7 or 8 weeks of age. A second group
of mice was maintained disease-free with 10 mg/kg of anti-TNF-α mAb weekly starting
at 4 weeks of age; when they reached 24 weeks of age, the treatment was stopped and
these 'aged mice' were allowed to develop arthritis before they were enrolled into
the study. The enrollment criteria for both young and aged mice required them to have
a clinical score of at least 2 on two limbs. The experimental protocols were approved
by Centocor's Institutional Animal Care and Use Committee.

The murine anti-TNF-α mAb used in this study binds with high affinity to hTNF-α and
prevents binding to the TNF receptor [10]. Anti-TNF-α mAb was administered by intraperitoneal injection at 10 mg/kg weekly
and the animals in the control group received weekly injections of saline.

Clinical scores

Clinical scores were based on a previously described scoring system [11] modified as follows: 0 (normal), 1 (edema or distortion of paw or ankle joints),
2 (distortion of paw and ankle joints), or 3 (ankylosis of wrist or ankle joints).
The sum of all four paws was scored weekly, with a maximum possible score of 12 per
mouse.

Histologic processing of joints

Specimens were processed and scored as described previously [12,13] and in the supplementary material.

ELISA assay

Serum samples were separated from whole blood, which was collected by intracardiac
puncture at baseline and at 3, 6, and 16 weeks post-treatment. Serum levels of hTNF-α
were determined by ELISA at a 1:2 dilution according to the manufacturer's instructions
(Biosource International, Camarillo, CA, USA).

RNA isolation and RNase protection assay

Samples were prepared as described in the Supplementary material and analyzed in an
RNase protection assay according to the manufacturer's instructions (PharMingen, San
Diego, CA, USA).

Statistical analysis

All data are expressed as means ± standard deviation unless noted otherwise. Statistical
significance was tested using analysis of variance for multiple groups. If significant
differences were found, pair-wise testing was performed using Tukey's test. The level
of significance for all comparisons was set at P < 0.05.

Results

Amelioration of established polyarthritis by treatment with anti-TNF-α mAb

Increased cartilage turnover and repair in response to insult has been observed in
young mice, whereas aged mice show decreased cartilage turnover after closure of the
growth plate [14]. Therefore, anti-TNF-α treatment was evaluated in both young (7–8-week-old) and aged
(27–28-week-old) Tg197 mice with established arthritis. The mean clinical arthritis
score at baseline was 6 (Fig. 1), indicating arthritis was established at the commencement of treatment. The clinical
arthritis score increased progressively in the saline-treated group and these mice
were humanely killed at 6 weeks. In contrast, the clinical arthritic score was markedly
decreased in the groups, both young and aged, treated with anti-TNF-α, indicating
a marked suppression of their arthritic symptoms (Fig. 1a,1b). Mice in the saline-treated group progressively lost weight, whereas mice in the
anti-TNF-α group showed a significant weight gain throughout the study (see Supplementary
Fig. 1). The arthritic score (weeks 1 to 16 post-treatment; Fig. 1a,1b) and weight gain (weeks 5 to 16 post-treatment; Supplementary Fig. 1) in the group treated with anti-TNF-α were significantly improved in comparison with
the saline-treated group in both young and aged mice. More importantly, the arthritic
scores for the group treated with anti-TNF-α were significantly improved in comparison
with the baseline score from weeks 4 through 16 in both young and aged mice (Fig.
1a,1b). These results indicate that disease progression was not just prevented but was
reversed following anti-TNF-α treatment.

Figure 1. Treatment with anti-TNF-α monoclonal antibodies reverses established arthritis in
Tg197 mice. (a) Young mice (at 7 to 8 weeks of age) or (b) aged mice (at 27 to 28 weeks of age) with established arthritis were randomized to
three groups to be humanely killed immediately (baseline, open square, n = 5) or to receive weekly doses of saline (open circle, n = 10) or 10 mg/kg of anti-TNF-α (closed square, n = 10 through 6 weeks of age and n = 5 from 7 to 16 weeks). The mice were monitored weekly for arthritic score. *P < 0.05, versus saline-treated controls; +P < 0.05, versus baseline score; both scores remaining significant until the end of
the study. Data are from two experiments. TNF, tumor necrosis factor.

Effects of treatment with anti-TNF-α mAb on joint histopathology

All joints were scored in a blinded fashion for synovitis, bone erosions, and cartilage
degradation using a predefined scoring system. The mean histologic scores for each
treatment group at various time points are shown in Fig. 2. Saline treatment was followed by increased histologic scores for synovitis, bone
erosions, and cartilage damage relative to that observed at baseline. Anti-TNF-α treatment
for 6 weeks significantly reduced all three histologic scores relative to the baseline
score in young mice (Fig. 2) and two of the three histologic scores in aged mice (Supplementary Fig. 2). After 16 weeks of anti-TNF-α treatment, synovial inflammation and bone erosions
were almost completely resolved in both young and aged mice, and a significant reduction
in cartilage degradation in comparison with baseline was maintained in young mice.
Cartilage degradation in aged mice treated with anti-TNF-α was similar to that at
the baseline measurement and significantly lower than that in the saline treatment
group (Supplementary Fig. 2).

Representative tissue sections of the ankle joints from young mice are shown in Fig.
3. Evidence of arthritic disease was readily observed at baseline (Fig. 3a,3b), and the disease continued to progress in saline-treated mice at 6 weeks, with increased
cell proliferation and inflammatory cell infiltration, erosion of cartilage and bone,
and loss of proteoglycan from the cartilage (Fig. 3c,3d). However, after 6 weeks of anti-TNF-α treatment, improvements in all histopathological
parameters were observed with both staining procedures (Fig. 3e,3f). No visible evidence of bone or cartilage erosion was observed and all signs of
cellular proliferation and infiltration disappeared. The proteoglycan content of the
cartilage was notably improved. Extended treatment with anti-TNF-α (for 16 weeks)
maintained normal joint architecture (data not shown). These results demonstrate that
blocking hTNF-α reverses joint inflammation and promotes joint healing in this model
of polyarthritis.

Figure 3. Histology shows significant healing of arthritic joints in young Tg197 mice after
treatment with anti-TNF-α mAb. Tissue sections of the ankle joints were obtained from
young Tg197 mice with established arthritis at baseline (a,b) and after 6 weeks of treatment with saline (c,d) or anti-TNF-α (e,f). Sections were stained with hematoxylin and eosin (a, c, e) or toluidine blue (b,
d, f). The arrows in panels (a) and (b) indicate areas of cell infiltration (1), erosions
(2) and loss of proteoglycan from cartilage (3). Original magnification, 20×. mAb,
monoclonal antibody; TNF, tumor necrosis factor.

Inhibition of TNF-α and other proinflammatory cytokines

To define the underlying mechanisms by which anti-TNF-α mAb ameliorated joint inflammation
and structural damage, the levels of various proinflammatory mediators were examined.
First, circulating hTNF-α levels as detected by ELISA were more than halved as early
as 3 weeks and the reduction was sustained for 16 weeks after treatment with anti-TNF-α
mAb in comparison with saline-treated animals (Fig. 4a). In addition, murine IL-6, IL-1β, and IFN-γ mRNA, which are expressed in the arthritic
joint tissues, were clearly diminished following anti-TNF-α treatment when total joint
RNA was examined by RNase protection assays (see Supplementary Fig. 3). When the levels of murine IL-6, IL-1β, and IFN-γ mRNA were standardized to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (Fig. 4b,4c,4d), a 2–3-fold reduction in murine IL-6 and IFN-γ was observed in the aged animals
treated with anti-TNF-α in comparison with those treated with saline at 6 weeks post-treatment
(Fig. 4b,4c). Interestingly, local expression of murine TNF-α mRNA was not affected by treatment
with anti-TNF-α antibodies, suggesting that hTNF-α plays a crucial role in the pathogenesis
of arthritis in this model.

Figure 4. Treatment with anti-TNF-α monoclonal antibodies inhibits proinflammatory cytokine
production in Tg197 mice. (a) Sera were collected from young animals at the indicated times. Levels of hTNF-α were
determined by ELISA and expressed as mean ± SEM of each group. (b-d) Detection of proinflammatory cytokines, murine IL-6, IFN-γ, and IL-1β mRNA in joint
tissue of aged mice. RNA was extracted from the joint tissues of the mice at baseline
and 6 weeks after treatment with either saline or anti-TNF-α mAb. An RNase protection
assay was then performed. The ratios of individual cytokine mRNA to GAPDH in animals
treated with anti-TNF-α mAb are compared with those in saline-treated animals or animals
at baseline. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mAb, monoclonal antibody;
TNF, tumor necrosis factor.

Supplementary Figure 1. Anti-TNF-α treatment reverses weight loss in arthritic animals. (a) Young (at 7 to 8 weeks of age) or (b) aged mice (at 27 to 28 weeks of age) with established arthritis (arthritic score
of 2 in at least two limbs) were randomized to three groups to be humanely killed
immediately (baseline, n = 5) or to receive weekly doses of saline (open circle, n = 10) or 10 mg/kg of anti-TNF-α (closed square, n = 10 through 6 weeks and n = 5 from 7 to 16 weeks). Weight gain was measured as change from pretreatment weight.
*P < 0.05, versus saline controls, and remained significant until the end of the study.
TNF, tumor necrosis factor.

Supplementary Figure 3. Detection of mRNA of proinflammatory cytokines murine TNF-α, IL-6, and IFN-γ in joint
tissue of aged mice. RNA was extracted from the paw joints at baseline and after 6
weeks of treatment with saline or anti-TNF-α. An RNase protection assay was then performed.
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; L32, ribosomal protein; TNF, tumor
necrosis factor.

Discussion

Currently, MTX is the most widely used disease-modifying antirheumatic drug for the
treatment of RA. However, MTX treatment rarely results in complete disease remission
[15] and only slows the progression of joint erosion [16]. In addition, long-term treatment with MTX is associated with pulmonary [17] and liver toxicities [18] and other side effects. Therefore, a treatment option that facilitates reversal of
joint damage and has longer effectiveness and fewer side effects is desirable.

Experimental arthritis models have contributed to the basic understanding of joint
disease and to the development of effective antiarthritic agents [19]. Several models have been used to mimic human RA, ranging from immunization with
cartilage components to infection with joint trophic organisms [20,21]. Blocking TNF-α [6,22] or IL-1 [23,24] in these models has routinely shown benefit, although some questions remain regarding
the role of these cytokines in mitigating joint inflammation versus preventing cartilage
degradation and bone erosion [25].

Using the Tg197 mice, we have shown that maintenance anti-TNF-α therapy, initiated
after joint inflammation and erosions have occurred, allows damaged joints to heal.
Specifically, anti-TNF-α mAb proved equally effective in reversing joint synovitis
and erosions both in young mice, where an active repair process to damage occurs,
and aged mice, where the repair process has relatively slowed. However, the repair
of cartilage damage was different between young and aged mice. Cartilage in young
mice treated with anti-TNF-α was significantly improved relative to saline-treated
and baseline mice. By comparison, treatment of aged mice with anti-TNF-α prevented
further cartilage damage but did not improve histological scores relative to baseline
values. Mechanistically, the therapeutic effect of anti-TNF-α appears to be due to
either neutralization of soluble hTNF-α or inhibition of hTNF-α production in the
diseased joint. Additionally, other proinflammatory cytokine mRNAs were decreased
in the local diseased tissues either through suppression of inflammatory cell infiltration
or inhibition of cytokine production. Moreover, anti-TNF-α treatment resulted in a
modest inhibition of murine IL-1β production in the diseased joint, which is consistent
with previous findings that anti-TNF-α antibody inhibits the generation of IL-1 in
collagen-induced arthritis and IL-1, IL-6, and IL-8 in rheumatoid synovial cultures
[26,27]. Our study provides preclinical evidence supporting the use of anti-TNF-α mAb in
ameliorating arthritic pathology.

The progressive arthritis observed in Tg197 mice is similar to the pathology in patients
with RA. Recent clinical data indicate that the blockade of TNF-α significantly reduces
the signs and symptoms of RA [28,29]and inhibits the progression of structural damage [30,31]. It remains to be seen whether extended anti-TNF-α therapy might permit regeneration
of articular cartilage and bone in established human disease, where multiple etiological
pathways may contribute to the RA disease syndrome [32,33]. Nevertheless, it is likely that much of the RA pathology involves TNF-α activation,
and the results from the Tg197 model provide a sound scientific rationale for the
therapeutic benefits observed following anti-TNF-α treatment in RA patients.

Conclusion

Progression of established polyarthritis in the Tg197 hTNF-α transgenic mouse can
be reversed by treatment with anti-TNF-α mAb, as shown by significant improvement
in clinical and histological scores.

Supplementary material

Supplementary materials and methods

Histologic processing of joints

Joint tissues from wrists, ankles, elbows, and knees were fixed in 10% buffered formalin
overnight, decalcified in 10% formic acid for 18 days, dehydrated, and then embedded
in paraffin. Specimens were cut longitudinally to the midline, and 5-m sections mounted
for staining with hematoxylin and eosin or toluidine blue. Joint sections stained
with hematoxylin and eosin were scored for synovitis and bone erosions, as described
elsewhere [12,13]. Sections stained with toluidine blue were scored for cartilage degradation (0, normal
staining; 1, some loss of staining; 2, moderate loss of staining; 3, weak staining;
4, very weak staining; 5, no visible staining). Specimens were examined using light
microscopy and scored in a blinded fashion with regard to treatment.

RNA isolation and RNase protection assay

Excised ankle and paw joints were immediately frozen in liquid nitrogen and processed
in a freezer/mill (SPEX Certi-Prep, Metuchen, NJ, USA) with liquid nitrogen at 2-minute
pulses. RNA was isolated from 50 mg of the joint powder with 1 ml Trizol reagent according
to the manufacturer's instructions (Gibco BRL, Grand Island, NY, USA). RNA was extracted
with 0.2 ml chloroform, precipitated with 0.5 ml isopropyl alcohol, and resuspended
in 30 l diethyl pyrocarbonate-treated water. Ten micrograms of total RNA from each
sample was used for RNase protection assay (mCK -2b and -3b Multi-Probe Template Sets,
PharMingen, San Diego, CA, USA), exposed to a Phosphor screen, and quantified by a
Phosphorimager with the use of Image Quant software (Molecular Dynamics, Sunnyvale,
CA, USA). The signal-intensity ratio of different cytokine messenger RNA (mRNA) to
GAPDH was determined.

Acknowledgments

The authors thank Dr George Kollias for providing the Tg197 transgenic breeder mice
and Dr Bernie Scallon for polymerase chain reaction analysis of the Tg197 mice.

Griffiths RJ: The use of animals in the search for anti-inflammatory drugs. In In Mechanisms and Models in Rheumatoid Arthritis.. Edited by Henderson B, Edwards JCW, Pettipher ER. London: Academic Press; 1995:527-537.